Study Notes: Measuring Molecular Weight

Remember that molecular weight is actually a ratio called relative molecular
mass (Mr).

Protein bands detected on the gel need to be identified. Identification
involves the following factors.

Measurement of the molecular weight (Mr)

Knowledge and experience with these particular samples and proteins

Immunological techniques such as western blotting to identify proteins
(Step 7)

Purification and analysis of the protein such as amino acid sequencing
and comparison with published protein sequence data

Other analytical techniques.

Measurement of the Mr is simple and can be carried
out on the gel. Remember that in SDS-PAGE the effect of the SDS is to
impart a uniform negative charge on the protein based on the size of the
protein in a linear way. That is the charge:mass ratios for all proteins
are the same. In this case relative mobilities of proteins is based solely
on the sieving action of the gel.

The simplest way to measure Mr is to add a set of standard
proteins of known Mr to one or more wells in the gel.
These are called molecular weight markers and can be sourced commercially
in ranges to suit the % gel being run. They also come pre-stained in a
range of colours (Rainbow Markers) if required.

The relative migration (from the top of the gel in cm) of each of the
marker proteins is plotted on a graph against Mr to
form a standard curve. The Mr of an unknown protein
can then be simply calculated from the migration of that protein on the
gel.

An example of a set of Low Range Mr Markers that come
mixed, pre-diluted and ready to use, is shown below.

Protein

Relative Molecular Mass
- Mr

Ovalbumin

43 kd

-chymotrypsinogen

25.7 kd

-lactoglobulin

18.4 kd

Lysozyme/Cytochrome C

13.3 kd (14.3 and 12.3)

Bovine Trypsin Inhibitor

6.2 kd

Insulin

3.0 kd

The standard curve plotted from SDS-PAGE on a 15% gel.

Calibration chart for Mr standards.

An example of the determination of the Mr
of an unknown protein Px is shown. Px was run on a 15% gel and migrated
9 cm into the gel. Using the standard curve the Mr
is calculated to be 9 kd.

An example of the use of Mr markers is given
below. This gel shows analyses of virus-infected cells run on an 11% to
24.4% polyacrylamide gradient gel. The Mr
markers are shown in the left hand lane and the Mr
of six of them is indicated on the right hand side of the gel. Notice
the ‘splaying’ of the sample lanes from top to bottom due
to the high strength acrylamide used towards the bottom of the gradient.

An SDS-PAGE separation on a gradient gel. Notice the Mr
markers in the far left lane.